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Ligand-dependent corepressor LCoR interacts with the progesterone receptor (PR) and estrogen receptor ERalpha in the presence of hormone. LCoR contains tandem N-terminal PXDLS motifs that recruit C-terminal-binding protein (CtBP) corepressors as well as a C-terminal helix-turn-helix (HTH) domain. Here, we analyzed the function of these domains in coregulation of PR- and ERalpha-regulated gene expression. LCoR and CtBP1 colocalize in nuclear bodies that also contain CtBP-interacting protein CtIP and polycomb group repressor complex marker BMI1. Coexpression of CtBP1 in MCF7 or T47D breast cancer cells augmented corepression by LCoR, whereas coexpression of CtIP did not, consistent with direct interaction of LCoR with CtBP1, but not CtIP. The N-terminal region containing the PXDLS motifs is necessary and sufficient for CTBP1 recruitment and essential for full corepression. However, LCoR function was also strongly dependent on the helix-turn-helix domain, as its deletion completely abolished corepression. LCoR, CtBP, and CtIP were recruited to endogenous PR- and ERalpha-stimulated genes in a hormone-dependent manner. Similarly, LCoR was recruited to estrogen-repressed genes, whereas hormone treatment reduced CtBP1 binding. Small interfering RNA-mediated knockdown of LCoR or CtBP1 augmented expression of progesterone- and estrogen-stimulated reporter genes as well as endogenous progesterone-stimulated target genes. In contrast, their ablation had gene-specific effects on ERalpha-regulated transcription that generally led to reduced gene expression. Taken together, these results show that multiple domains contribute to LCoR function. They also reveal a role for LCoR and CtBP1 as attenuators of progesterone-regulated transcription but suggest that LCoR and CtBP1 can act to enhance transcription of some genes.

Ligand-dependent corepressor LCoR was identified as a protein that interacts with the estrogen receptor alpha (ERalpha) ligand binding domain in a hormone-dependent manner. LCoR also interacts directly with histone deacetylase 3 (HDAC3) and HDAC6. Notably, HDAC6 has emerged as a marker of breast cancer prognosis. However, although HDAC3 is nuclear, HDAC6 is cytoplasmic in many cells. We found that HDAC6 is partially nuclear in estrogen-responsive MCF7 cells, colocalizes with LCoR, represses transactivation of estrogen-inducible reporter genes, and augments corepression by LCoR. In contrast, no repression was observed upon HDAC6 expression in COS7 cells, where it is exclusively cytoplasmic. LCoR binds to HDAC6 in vitro via a central domain, and repression by LCoR mutants lacking this domain was attenuated. Kinetic chromatin immunoprecipitation assays revealed hormone-dependent recruitment of LCoR to promoters of ERalpha-induced target genes in synchrony with ERalpha. HDAC6 was also recruited to these promoters, and repeat chromatin immunoprecipitation experiments confirmed the corecruitment of LCoR with ERalpha and with HDAC6. Remarkably, however, although we find evidence for corecruitment of LCoR and ERalpha on genes repressed by the receptor, LCoR and HDAC6 failed to coimmunoprecipitate, suggesting that they are part of distinct complexes on these genes. Although small interfering RNA-mediated knockdown of LCoR or HDAC6 augmented expression of an estrogen-sensitive reporter gene in MCF7 cells, unexpectedly their ablation led to reduced expression of some endogenous estrogen target genes. Taken together, these data establish that HDAC6 can function as a cofactor of LCoR but suggest that they may act in enhance expressing some target genes.

Both B-lymphoblastoid cell lines and tonsillar B lymphocytes express receptors for platelet-activating factor (PAF). In lymph node germinal centres, B lymphocytes interact with follicular dendritic cells (FDCs), which present antigen-containing immune complexes to B lymphocytes. FDCs have phenotypic features that are similar to those of stromal cells and monocytes and may therefore be a source of lipid mediators. In this study, we evaluated the effects of the PAF antagonist WEB 2170 on the activation of tonsillar B lymphocytes by FDCs. FDCs were isolated from tonsils by Bovine Serum Albumin (BSA) gradient centrifugation. After being cultured for 6 to 10 days, they were incubated with freshly isolated B cells in the presence or absence of the specific PAF receptor antagonist WEB 2170. B-lymphocyte proliferation was assessed by [3H]-thymidine incorporation, and immunoglobulin (Ig) G and IgM secretion was assessed by enzyme-linked immunosorbent assay (ELISA). WEB 2170 (10-6 to 10-8 M) inhibited [3H]-thymidine incorporation by up to 35% +/- 3%. Moreover, the secretion of IgG and IgM was inhibited by up to 50% by WEB 2170 concentrations ranging from 10-6 to 10-8 M. There was no evidence of toxicity by trypan blue staining, and the addition of WEB 2170 to B cells in the absence of FDCs did not inhibit the spontaneous production of IgG or IgM. The effect of the PAF antagonist is primarily on B lymphocytes, as reverse transcription polymerase chain reaction detected little PAF receptor messenger ribonucleic acid (mRNA) from FDCs. These data suggest that endogenous production of PAF may be important in the interaction of B lymphocytes with FDCs.

LCoR (ligand-dependent corepressor) is a transcriptional corepressor widely expressed in fetal and adult tissues that is recruited to agonist-bound nuclear receptors through a single LXXLL motif. LCoR binding to estrogen receptor alpha depends in part on residues in the coactivator binding pocket distinct from those bound by TIF-2. Repression by LCoR is abolished by histone deacetylase inhibitor trichostatin A in a receptor-dependent fashion, indicating HDAC-dependent and -independent modes of action. LCoR binds directly to specific HDACs in vitro and in vivo. Moreover, LCoR functions by recruiting C-terminal binding protein corepressors through two consensus binding motifs and colocalizes with CtBPs in the nucleus. LCoR represents a class of corepressor that attenuates agonist-activated nuclear receptor signaling by multiple mechanisms.

The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is key mediator of calcium homeostasis and is a component of the complex homeostatic system of the skin. 1,25-(OH)2D3 regulates cellular differentiation and proliferation and has broad potential as an anticancer agent. Oligonucleotide microarrays were used to assess profiles of target gene regulation at several points over a 48 h period by the low calcemic 1,25-(OH)2D3 analog EB1089 in human SCC25 head and neck squamous carcinoma cells. One hundred fifty-two targets were identified, composed of 89 up- and 63 down-regulated genes distributed in multiple profiles of regulation. Results are consistent with EB1089 driving SCC25 cells toward a less malignant phenotype, similar to that of basal keratinocytes. Targets identified control inter- and intra-cellular signaling, G protein-coupled receptor function, intracellular redox balance, cell adhesion, and extracellular matrix composition, cell cycle progression, steroid metabolism, and more than 20 genes modulating immune system function. The data indicate that EB1089 performs three key functions of a cancer chemoprevention agent; it is antiproliferative, it induces cellular differentiation, and has potential genoprotective effects. While no evidence was found for gene-specific differences in efficacy of 1,25-(OH)2D3 and EB1089, gene regulation by 1,25-(OH)2D3 was generally more transient. Treatment of cells with 1,25-(OH)2D3 and the cytochrome P450 inhibitor ketoconazole produced profiles of regulation essentially identical to those observed with EB1089 alone, indicating that the more sustained regulation by EB1089 was due to its resistance to inactivation by induced 24-hydroxylase activity. This suggests that differences in action of the two compounds arise more from their relative sensitivities to metabolism than from differing effects on VDR function.

Analogs of 1alpha,25-dihydroxyvitamin D(3) (1alpha, 25(OH)2D3) inhibit growth in vitro and in vivo of cells derived from a variety of tumors. Here, we examined the effects of 1alpha,25(OH)2D3 and its analog EB1089 on proliferation and target gene regulation of human head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15, and SCC25. A range of sensitivities to 1alpha,25(OH)2D3 and EB1089 was observed, from complete G0/G1 arrest of SCC25 cells to only 50% inhibition of SCC9 cell growth. All lines expressed similar levels of vitamin D3 receptor (VDR) mRNA and protein, and no significant variation was observed in 1alpha,25(OH)2D3-dependent induction of the endogenous 24-hydroxylase gene, or of a transiently transfected 1alpha,25(OH)2D3-sensitive reporter gene. The antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC25 cells were analyzed by screening more than 4,500 genes on two cDNA microarrays, yielding 38 up-regulated targets, including adhesion molecules, growth factors, kinases, and transcription factors. Genes encoding factors implicated in cell cycle regulation were induced, including the growth arrest and DNA damage gene, gadd45alpha, and the serum- and glucocorticoid-inducible kinase gene, sgk. Induction of GADD45alpha protein in EB1089-treated cells was confirmed by Western blotting. Moreover, while expression of proliferating cell nuclear antigen (PCNA) was reduced in EB1089-treated cells, coimmunoprecipitation studies revealed increased association between GADD45alpha and PCNA in treated cells, consistent with the capacity of GADD45alpha to stimulate DNA repair. While 1alpha,25(OH)2D3 and EB1089 modestly induced transcripts encoding the cyclin-dependent kinase inhibitor p21(waf1/cip1), no changes in protein levels were observed, indicating that p21(waf1/cip1) induction does not contribute to the antiproliferative effects of 1alpha,25(OH)2D3 and EB1089 in SCC cells. Finally, in partially resistant SCC9 cells, there was extensive loss of target gene regulation (10 of 10 genes tested), indicating that resistance arises from widespread loss of 1alpha,25(OH)2D3-dependent gene regulation in the presence of normal levels of functional VDRs.

Background: 1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its analogues inhibit growth of various types of cancer cells. Although the therapeutic potential of 1,25(OH)(2)D(3) is limited by its tendency to induce hypercalcemia, analogues such as EB1089 are potent inhibitors of cell growth and exhibit reduced calcemic effects. We analyzed the antiproliferative and calcemic effects of EB1089 in tissue culture and animal models of head and neck squamous cell carcinoma (SCC) to investigate its potential as a chemotherapeutic/chemopreventive agent., Methods: The effects of 1,25(OH)(2)D(3) and EB1089 on cell growth and expression of p21(WAF1/CIP1) and p27(KIP1), which encode cyclin-dependent kinase inhibitors, and a novel target, gadd45alpha, a growth-arrest and DNA-damage gene, were monitored in cultured murine AT-84 SCC cells. The effects of these agents on AT-84 cell growth in vitro and on growth of AT-84 tumors in syngeneic C3H mice were monitored; treatment started at the time of tumor implantation (early tumor model) or after 12 days (late tumor model). Weight and serum calcium levels were also monitored in these animals. All P values were two-sided., Results: Both 1,25(OH)(2)D(3) and EB1089 arrested proliferation of AT-84 cells in G(0)/G(1) phase, inhibited p21(WAF1/CIP1) expression, and induced expression of p27(KIP1) protein. 1,25(OH)(2)D(3) also enhanced the expression of gadd45alpha, apparently by a p53-independent mechanism. There was a statistically significant decrease in tumor growth for 1,25(OH)(2)D(3)-treated mice (P<.001 for early tumor model) and EB1089-treated mice (P<.001 and P =.001 for early and late tumor models, respectively). Unlike 1,25(OH)(2)D(3), EB1089 did not induce cachexia or hypercalcemia. The effects of 1,25(OH)(2)D(3) and EB1089 on expression of p21(WAF1/CIP1) and GADD45alpha were similar in tumors and in vitro., Conclusions: EB1089 completely inhibited growth of AT-84 SCC cells at nanomolar concentrations, reduced tumor growth, and did not have calcemic effects. Our results support continued investigation of EB1089 as a chemopreventive/chemotherapeutic agent for head and neck SCC.

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)2D3] inhibits growth of cells derived from a variety of tumors in vitro and in vivo. Proliferation in vitro of human SCC25 cells, derived from a primary squamous cell carcinoma (SCC) of the tongue, was blocked by 1,25(OH)2D3 and its analog EB1089. A similar effect was observed with 13-cis retinoic acid (RA), which has been used in chemoprevention of SCC. We identified amphiregulin, a member of the epidermal growth factor family, as a 1,25(OH)2D3 target gene in SCC25 cells. Induction of amphiregulin mRNA by 1,25(OH)2D3 was rapid and sustained over 48 h, and was unaffected by cycloheximide. 1,25(OH)2D3 also induced amphiregulin mRNA in estrogen receptor-positive and -negative human breast cancer cell lines, but not in LNCaP human prostate cancer cells. RAR- or RXR-specific retinoids did not affect amphiregulin mRNA levels in SCC25 cells; however, 13-cis RA partially blocked the response to 1,25(OH)2D3. Amphiregulin partially inhibited growth of SCC25 cells in culture. Our data show that amphiregulin is a 1,25(OH)2D3 target gene, and suggest that its induction may contribute to the growth inhibitory effects of 1,25(OH)2D3., (Copyright 2001 Academic Press.)

Platelet-activating factor receptor (PAFR) has been identified in B cell lines and primary human B cells, but the regulation of PAFR during B cell activation has not been completely elucidated. In the present study, we have investigated the effects of B cell activation on PAFR binding parameters, PAFR mRNA and PAF-triggered intracellular calcium mobilization. The human B lymphoid cell line LA350 was shown to exhibit high levels of PAFR (48,550 +/- 4,310 sites/cell) as determined by radio-ligand binding assay with PAFR antagonist [3H]WEB2086. Treatment with phorbol 12,13-dibutyrate caused a biphasic reduction of PAFR binding. The early phase was inhibited by the protein kinase C inhibitor bisindolylmaleimide I (BIM), whereas the late phase was not blocked by BIM, protein tyrosine kinase inhibitor genistein, or the mitogen-activated protein kinase/extracellular signal-related kinase inhibitor PD98059. However, staurosporine, a broad-spectrum protein kinase inhibitor, completely inhibited the late phase down-regulation. Ionomycin also decreased [3H]WEB2086 binding sites, whereas the combination of PDB and ionomycin induced a greater reduction than either agent alone. Cross-linking of B cell receptor by anti-IgM Ab also induced down-regulation of PAFR, which was abolished by genistein or PD98059, but not by BIM or staurosporine. The decrease in surface PAFR number was closely paralleled by the reduction in PAFR mRNA both in LA350 cells and human tonsillar B cells, and was associated with decreased response to PAF indicated by decreased intracellular calcium mobilization. These data show that multiple signaling pathways are involved in down-regulating PAFR expression during B cell activation and development.

Although platelet-activating factor (PAF) receptors have been found on B lymphoblastoid cell lines, the action of PAF on freshly isolated human B cells has not been clearly demonstrated. Using a sensitive semiquantitative reverse-transcriptase PCR, we have found PAF receptor mRNA expressed by tonsillar B lymphocytes, but little in T lymphocytes. Examination of Percoll-fractionated tonsillar B cells indicated that the low density (primarily germinal center cells) and medium density fractions had approximately twofold more PAF receptor mRNA relative to the high density fraction. PAF (10-7 M) stimulated increases in intracellular Ca2+ that were consistently higher in the low and medium density B lymphocytes compared with high density cells. The PAF receptor antagonist Web 2170 inhibited this. Addition of PAF, but not lyso- or enantio-PAF, induced four- to sixfold greater synthesis of IgM and IgG in low and medium density cells compared with unstimulated controls, but had little effect on Ig production by high density cells. To investigate how PAF may influence Ig synthesis, PAF-stimulated B cells were examined for production of the Th2-type cytokines IL-4 and IL-13. PAF induced IL-4 and IL-13 mRNA expression in 17% of CD20+ cells, and IL-4 was detected in cell supernatants after 48-72 h of culture. Together, these data strongly suggest that functional PAF receptors are expressed on B cells in tonsils.

B lymphocyte development is characterized by deletion via apoptosis of immature cells that are insufficiently stimulated. We have previously demonstrated that crosslinking of the B cell receptor (BCR) using anti-IgM antibodies (alphaIgM) (2 microg/ml) in Ramos B lymphoblastoid cells causes deletion of 30-40% of cells by apoptosis in 24 h. Addition of the potent lipid mediator platelet-activating factor (10(-7) M) to alphaIgM stimulated Ramos cells significantly decreases the number of apoptotic cells as measured by annexin V labeling. We have characterized the phenotype of Ramos cells that have not become apoptotic following BCR stimulation. In these cells, there is a significant decrease in the surface expression of the VLA-4 adhesion molecule (31% of control expression) and surface IgM expression (sIgM) (53% of control expression). Significantly fewer cells co-incubated with platelet-activating factor (PAF) underwent apoptosis, and the remaining cells maintained control levels of VLA-4 (104% of control expression) and sIgM expression (104% of control). All of these protective effects were inhibited by the specific PAF receptor antagonist, WEB 2170. The action of PAF on alphaIgM induced apoptosis was not inhibited by either cycloheximide or cytochalasin B, suggesting that de novo protein synthesis and F-actin polymerization were not implicated in the rescue of Ramos cells by PAF. In contrast, the ability of PAF to maintain sIgM and VLA-4 expression at control levels was inhibited by cycloheximide (7. 5 microg/ml). Cytochalasin B (5 microg/ml) had no effect on sIgM expression but blocked the decrease in VLA-4 expression mediated by alphaIgM. These data indicate that PAF's effect on rescuing and maintaining alphaIgM stimulated Ramos B cells is mediated via at least two pathways. Abrogation of apoptosis does not require de novo protein synthesis, while maintenance of sIgM and VLA-4 expression requires protein synthesis., (Copyright 1999 Academic Press.)

Background: Platelet activating-factor (PAF), an ether-linked phospholipid, is a potent activator of B lymphocyte cell lines. The related ester-linked phospholipid, 1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine (PAGPC), is synthesized by tissues important in B-cell development., Objectives: We examined whether PAGPC was capable of influencing immunoglobulin synthesis in B lymphocytes and compared its action with that of PAF. We also examined the interaction of the two mediators as agonists or competitive antagonists., Methods: Ramos, an IgM-secreting immature B-cell line that expresses PAF receptor, was used in these experiments. The effect of PAF, PAGPC, or both mediators together on IgM secretion and anti-IgM-mediated apoptosis was measured., Results: Both PAF and PAGPC stimulated IgM production in Ramos cells in a dose-dependent fashion, with PAGPC being approximately three logs less potent than PAF. The effect of both mediators was inhibited by specific PAF receptor antagonists. Preincubation with suboptimal concentrations of PAGPC inhibited the ability of PAF to increase IgM secretion by Ramos cells. Additionally, preincubation with low concentrations of PAGPC prevented PAF from rescuing Ramos cells from apoptosis induced by cross-linking the B-cell receptor with anti-IgM antibodies. PAGPC caused PAF receptor desensitization because displacement of bound PAGPC with high concentrations of bovine serum albumin did not reverse its PAF antagonist effect., Conclusions: PAF and PAGPC are biologically active phospholipids, but PAF is approximately 1000 times more potent. At high concentrations, PAGPC acts similarly to PAF, whereas at lower concentrations, PAGPC acts as a functional PAF antagonist. Because it is secreted at sites of inflammation and allergic reactions, PAGPC may be an endogenous regulator of the effects of PAF.

B lymphocyte development is characterized by deletion, via apoptosis, of immature cells that are stimulated via the B cell receptor in the absence of a second signal. We have investigated whether platelet-activating factor (PAF), a potent B lymphocyte activator, can provide a complementary signal with B cell receptor ligation to abrogate apoptosis. Cross-linking of the surface IgM on Ramos B lymphoblastoid cells using anti-IgM Abs (2 microg/ml) caused programmed cell death in 34 +/- 5.4% of the cells. Coincubation of PAF (10(-7)M) with alphaIgM led to a significant decrease in apoptotic cells as measured by DNA laddering and TUNEL assay (13.8 +/- 3%). The effect of PAF was dose dependent (10(-7)-10(-9) M) and was inhibited by the specific PAF receptor antagonist, WEB 2170. PAF protected cells from the effect of alphaIgM for up to 1 h after it was added. alphaIgM-induced programmed cell death in Ramos cells was blocked by catalase and, therefore, is caused in part by the production of toxic hydroxyl radicals from hydrogen peroxide. We investigated the action of PAF on markers of intracellular oxidation. H2O2 in low doses induced apoptosis, via production of OH. radicals. PAF inhibited H2O2-induced apoptosis in Ramos cells; it also attenuated H2O2- and alphaIgM-mediated increases in hydroxyl radical (OH.) as measured by the oxidation of 2',7'-dichlorofluorescein diacetate to 2',7'-dichlorofluorescein and blocked the depletion of reduced glutathione induced by alphaIgM. PAF maintained IgM secretion, which was greatly inhibited by incubation with alphaIgM alone. These data indicate that PAF potentially provides an important cosignal to surface IgM-stimulated Ramos cells by inhibiting apoptosis. This is in part due to the activity of PAF in the oxidant/ antioxidant pathway.

The platelet activating factor receptor (hPAFR) is a GTP binding protein linked receptor of the rhodopsin family. The hPAFR gene maps to chromosome 1 and is characterized by the absence of introns in its coding region. Because PAF demonstrates unique properties as a growth factor in human B lymphoblastoid cell lines, we developed an RT-PCR in order to detect the presence of hPAFR mRNA. We examined the presence of the hPAFR in a series of B lymphoblastoid cell lines, as well as on fresh human B cells and the T-leukemia cell line Jurkat. All cells of B lineage expressed hPAFR mRNA, including a B cell line not previously shown to express functional hPAFR. In contrast, the T-leukemia cell line Jurkat expressed very low levels of hPAFR mRNA. We discuss the methodology and its validation in developing an RT-PCR for intronless genes such as the hPAFR.

Using C57BL/HPB mouse erythrocytes, hemagglutination has been observed with the Los Angeles and Colorado-1 strains of bovine herpesvirus type 1 and with 12 other Canadian field isolates as well. The specificity of the hemagglutination observed with the viral strains has been confirmed by a hemagglutination-inhibition assay.

The intracellular and structural polypeptides of the Los Angeles and Cooper 1 reference strains of bovine herpesvirus 1, together with 12 other Canadian field isolates, were analyzed by polyacrylamide gel electrophoresis. Although a few minor differences were noted among some isolates in regard to intracellular viral protein content, analysis of partly purified virus showed strikingly similar polypeptide profiles among 19 proteins with molecular masses of 14 to 145 kilodaltons (kDa). Moreover, a neutralizing monoclonal antibody produced against the Cooper 1 strain also neutralized all of the other 13 strains tested in this study and immunoprecipitated the major 90-kDa glycoprotein. A second monoclonal antibody with a high hemagglutination inhibition titer prevented hemagglutination of other strains tested and also reacted against the 90-kDa glycoprotein by immunoprecipitation, indicating that this glycoprotein is responsible for the hemagglutinating activity of the viral particle and carries an important neutralization epitope.